Enzymes that use DNA as a substract - DNA Polymerase I

Figure 1 - Functional domains in the Klenow Fragment (left)
and DNA Polymerase I (right).

DNA polymerase I is an enzyme that participates in the DNA replication of prokaryotes. It is the predominant and most abundant polymerizing enzyme found in E.coli. It was the first DNA polymerase to be identified and discovered by Arthur Kornberg, an American biochemist in 1956. He was able to characterize the enzyme initially in E.coli. In E.coli the enzyme is a single polypeptide chain comprised of 928 aminoacids and it has a molecular weight of 109kDa. Nevertheless, the enzyme does not occur only in E.coli but also in many prokaryotes. In prokaryotes it is mostly encoded by polA gene.

Uses:

It has three distinct functions: 3′ to 5′ exonuclease or “proofreading” function because it can remove a nucleotide from the 3’ terminus which was incorrectly inserted in the DNA strand, 5′ to 3′ exonuclease and 5′ to 3′ polymerase.

The second function is the 5’ to 3’ exonuclease. This enzyme has the ability to remove nucleotides from the 5’ terminus. Furthermore, the nucleotides removed can be either of the deoxyribonucleotides or ribonucleotides. The main goal of the 5′ to 3′ exonuclease activity is to remove ribonucleotide primers that are used in DNA replication.

The 5’ to 3’ polymerase is the main activity of the enzyme and it means that it can add nucleotides to the 3’-OH terminus of the previous nucleotide in a DNA template. In order for polymerase to start adding nucleotides it needs a primer (consisted of approximately 10 ribonucleotides) to begin DNA synthesis. DNA pol I adds approximately 15-20 nucleotides per second.

Common reaction buffer:

A common reaction buffer is Tris-HCl (pH 7.2).

References:

ScienceDirect, DNA Polymerases,, viewed in November 13, 2018, https://www.sciencedirect.com/topics/neuroscience/dna-polymerase-i

Worthington Biochemical Corporation, DNA Polymerase I, viewed in November 13, 2018, http://www.worthington-biochem.com/DNAECI/default.html

Biology Online, DNA Polymerase I, viewed in November 13, 2018, https://www.biology-online.org/dictionary/DNA_polymerase_I

Technical information of kit M205A, DNA polymerase I, Promega Corporation.

Evangelia Nikou (e8749) | Hugo Rodrigues (a85946) | Mélanie Pereira (a83980) 

Pedro Gonçalves (a84784) | Ricardo Fernandes (a86254)

University of Minho | School of Sciences | Department of Biology 
Degree in Applied Biology | Genes and Genomes